This invention relates to multi-functional chimeric proteins and the method of making these chimeric proteins by genetic recombinant techniques. The chimeric proteins of the present invention contain a cellulose binding domain.
Serum is a necessary supplement in an anchorage cell culture. It is used to help culture cells attach onto the culture plates as well as to enhance cell-growth. Serum is also the most expensive component in a culture medium primarily due to limited sources. However, the qualities of serum from different sources vary highly, which may directly or indirectly contribute to the unstable physiologic property of cells. Therefore, it is necessary to develop an economic substitute for serum while still enabling the stable physiologic property of cells.
Chimeric proteins, i.e., the proteins containing both a functional domain (catalytic or otherwise) and a binding domain, have been used in various ways, especially in protein purification. For example, the chimeric protein composed of a desired protein fused with the c-terminal of a glutathione S-transferase is used widely in bio-technologic related fields, wherein the fused protein can be purified by a glutathione-Sepharose column. By means of the association between the binding domain of the chimeric protein and the substrate of the binding domain which is immobilized on a solid matrix such as beads, resins, plates, etc, the desired products can be conveniently purified.
Cellulose, a major component of the cellular walls of plants, is a continuous linear glucose xcex2-1,4 linkage polysaccharide which is readily available in the nature. Cellulase is a hydrolase of cellulose, wherein the cellulase can digest the cellulose by cleaving the xcex2-1,4 glycosidic bonds of cellulose. It is well known that the sequence encoding the cellulase comprises a cellulose-binding domain (CBD) allowing cellulose to bind to cellulose and subsequently cleave the xcex2-1,4 glycosidic bonds of cellulose. It is disclosed in U.S. Pat. No. 5,496,934 that the CBD has a high affinity for crystalline cellulose having a Kd ranging from 1.5 to about 0.8, preferably from about 1.4 to about 0.8, and the chimeric protein comprising CBD and a second protein retains the avid binding capacity of the CBD to cellulose. By means of the binding affinity between the cellulose-binding domain and the cellulose, the cellulase can be immobilized on the matrix coated with cellulose by the binding domain thereof. In addition, the cellulose prices are 100-500 fold lower than those of glutathione-Sepharose, making cellulose an attractive, inexpensive matrix that can be used safely in food and pharmaceutical industries. Therefore, it is highly beneficial to develop a cheap purification system by joining the desired protein with a cellulose binding domain, or to create a substitute for serum by joining an anchorage enhancer and a growth factor with a cellulose binding domain.
Dead plants and fallen leaves in nature are digested by microorganisms with the ability of cellulose digestion. The microorganisms with the ability of cellulose digestion comprise eukaryotes such as eumycetes, and prokaryotes such as bacteria. The microorganisms described above can synthesize cellulase to digest the cellulose into small molecules, which can be further digested by other saccharide hydrolases. Gene cloning and sequencing results (Shoseyov et al., Proc. Natl. Acad. Sci. USA, 89:3483-3487, 1992) have demonstrated that the cellulase contains two independent functional regions: a catalytic domain with cellulase property, and a cellulose binding domain (CBD). Also, the biochemistry test results demonstrate that these two domains are functioned independently.
Chimeric proteins with a cellulose binding protein have been disclosed in several U.S. patents and literature. For example, U.S. Pat. No. 5,202,247 discloses a cellulose binding fusion protein having a substrate binding region of cellulose; U.S. Pat. No. 5,137,819 discloses cellulose binding fusion proteins for immobilization and purification of polypeptides; U.S. Pat. No. 5,340,731 describes a method of preparing a xcex2-1,4-glycan matrix containing a bound fusion protein; and U.S. Pat. No. 5,496,934 discloses nucleic acids encoding a cellulose binding domain. Moreover, Wierzba et al., Biotechnol. Bioeng. 47:147-154, 1995, discloses a chimeric protein which consists of the c-terminal binding domain of cellulase from bacteria and an amino acid sequence with the ability of cell-attachment. The chimeric protein can enhance cells anchorage on the matrix coated with cellulose.
The cellulose binding domain of the above patents and literature is primarily obtained from bacteria, wherein the cellulose binding domain located at the N-terminal of cellulase and the C-terminal catalytic domain are isolated from a proline- and threinine-rich amino acid sequence.
However, the cellulose binding domain of eumycetes is much shorter than that of bacteria, and the structure of the cellulose binding domain of eumycetes is much denser than that of bacteria.
The present invention involves the production of a recombinant chimeric protein which contains a cellulose binding domain from eumycetes. This chimeric protein not only allows the insertion of long amino acid sequences, but also is capable of allowing insertion of short amino acid sequence (e.g., three amino acid sequences). The chimeric protein is obtained by joining functional amino acid sequences on the N- and C-terminals of a cellulose binding domain. For example, one terminal of the cellulase binding domain can be joined to a cell-attachment enhancer, and the other terminal of the cellulase can be joined to a growth factor, thereby producing a chimeric protein that can enhance both the cells ability to anchor on the matrix coated with cellulose and cell growth. One difficulty that must be overcome, however, is how to correctly link the two disulfide bonds within the cellulose binding domain. Another difficulty is how the disulfide binds can be formed correctly and be exposed on the surface of the chimeric protein to retain the cellulose binding ability when the N-terminal cellulose binding domain is located between two amino acid sequences.
The invention features a multi-functional chimeric protein produced by genetic recombinant techniques, wherein two exogenous bifunctional amino acid sequences are respectively joined at the C-terminal and the N-terminal of the cellulose binding domain of cellulase obtained from Trichoderma konigii G39 (cellulobiohydrolase I, CEH I) to generate a recombinant protein. In addition to the cellulose binding ability, the function of the chimeric protein is determined by the sequences joined at the C-terminal and the N-terminal. By means of the cellulose binding ability of a cellulose binding domain (CBD), the desired protein joined with the CBD can be immobilized on the matrix, applied to cell cultures, as well as used for antibody or antigen detection and other medically related industries. The desired protein can be selected from the group consisting of thioredoxin, an Arginine-Glutamate-Asparate (RGD) tripeptide, Protein A, Protein G, streptavidin, avidin, Taq polymerase, non-Taq polymerase, alkaline phosphatase, RNase, DNase, restriction enzymes, peroxidases, glucanases, chitinases, beta and alpha glucosidases, beta and alpha glucoronidase, amylases, transferases, beta-lactamase, non-beta lactamase antibiotic modifying and degrading enzymes, luciferase, esterases, lipases, proteases, bacteriocines, antibiotics, enzyme inhibitors, growth factors, hormones, receptors, antigens, membrane proteins, nuclear proteins, transcriptional and translational factors and nucleic acid modifying enzymes.
This invention also provides a method of overexpressing a CBD fused product, wherein the method comprises the following steps: (a) providing a first DNA fragment comprising the coding sequence of the CBD; (b) joining a second DNA sequence and a third DNA sequence encoding two desired proteins to a 5xe2x80x2-end and a 3xe2x80x2-end of said first DNA fragment to form a recombinant DNA molecule encoding the CBD fused product; (c) cloning the recombinant DNA molecule in a vector with a selected marker to construct an expression vector comprising the recombinant DNA molecule encoding the CBD fused product; (d) transferring the expression vector into a cell to form a transformed cell; (e) overexpressing the CBD fused product in the transformed cell. The recombinant DNA molecule is preferably inserted into the vector downstream from a promoter site. The perferable promoter is T7.
The cellulose binding domain described above can be isolated from microorganisms such as the cellulobiohydrolase I of Trichoderma konigii G39 (CCRC 930014). Moreover, the first DNA fragment which encodes the cellulose binding domain can be modified by replacing the code encoding the fifth amino acid residue of the cellulose binding domain from tyrosine to tryptophan. The desired protein can be selected from the group consisting of thioredoxin, an Arginine-Glutamate-Asparate (RGD) tripeptide, Protein A, Protein G, streptavidin, avidin, Taq polymerase, non-Taq polymerase, alkaline phosphatase, RNase, DNase, restriction enzymes, peroxidases, glucanases, chitinases, beta and alpha glucosidases, beta and alpha glucoronidase, amylases, transferases, beta-lactamase, non-beta lactamase antibiotic modifying and degrading enzymes, luciferase, esterases, lipases, proteases, bacteriocins, antibiotics, enzyme inhibitors, growth factors, hormones, receptors, antigens, membrane proteins, nuclear proteins, transcriptional and translational factors and nucleic acid modifying enzymes. The cell for transformation can be either a prokaryote or a eukaryote. In addition, the method further comprises a step to purify the CBD fused product by an ion-exchange liquid chromatography such as an anion-exchange resin DE52.
Other features or advantages of the present invention will be apparent from the following drawings and detailed description of the example, and also the claims.